Thermo Scientific™ Eco57I (AcuI) (5 U/μL)

Description

Lambda DNA digested with Eco57I (AcuI), 1% agarose, 40 cleavage sites

conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.

5'...C T G A A G (N)₁₆▵...3'

3'...G A C T T C (N)₁₄▵...5'

Conditions for 100% Activity:

• [1X Buffer G] + SAM:[10mM Tris-HCl (pH 7.5 at 37°C), 10mM MgCl₂, 50mM NaCl and 0.1mg/mL BSA] + 0.01mM S-adenosylmethionine
• Incubate at 37°C

Storage Buffer:

• Eco57I is supplied in:10mM potassium phosphate (pH 7.4 at 25°C), 100mM NaCl, 1mM EDTA, 1mM DTT and 50% (v/v) glycerol

Ligation and Recleavage:

• After 10-fold overdigestion with Eco57I, approximately 70% of the DNA fragments can be ligated, but none of these can be recut due to the methylation of the recognition sequence by this enzyme

Methylation Effects:

• Dam: never overlaps-no effect
• Dcm: never overlaps-no effect
• CpG: never overlaps-no effect
• EcoKI: never overlaps-no effect
• EcoBI: may overlap-effect not determined

Note:

Eco57I requires only Mg² + for its activity, but is stimulated by S-adenosylmethionine. 0.01mM S-adenosylmethionine gives a 100-fold increase in Eco57I activity. Still, complete cleavage of some substrates with Eco57I is difficult to achieve. Eco57I concentration is determined by the maximum cleavage level achieved when no change in the fragmentation pattern is observed with further enzyme increase. Low salt, high glycerol (>5%) concentrations, pH >8.0 or a large excess of enzyme may result in star activity. Eco57I may remain associated with the cleaved DNA. This may cause DNA band shifting during electrophoresis. To avoid atypical DNA band patterns, use the 6X DNA Loading Dye & SDS Solution for sample preparation or heat the digested DNA in the presence of SDS prior to electrophoresis.